Type 1 allergies have dramatically increased worldwide in recent decades. Up to 20% of the population in industrialised countries suffer from complaints, such as allergic rhinitis, conjunctivitis or bronchial asthma, which are caused by allergens present in the air (aeroallergens), which are released by various sources, such as plant pollen, mites, mammals (cats, dogs, horses) and mould fungi. Severe allergies can also be initiated by insect stings or bites, such as, for example, of bees and wasps.
The type 1 allergy-initiating substances are proteins, glycoproteins or polypeptides. These allergens react via the mucous membranes after ingestion or with the IgE antibodies bound to the surface of mast cells in sensitised people after stings or bites. If two or more IgE antibodies are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in initiation of the allergic symptoms.
Birch pollen are the most frequent initiators of allergic reactions amongst tree pollen (Jarolim E. et al., 1989, Allergy 44:385-95). More than 90% of sufferers from birch pollen allergies have IgE antibodies against the major allergen Bet v 1 (Elfman, L. et al., 1997, Int. Arch. Allergy Immunol., 113: 249-51).
With the aid of cDNA sequences, it is possible to prepare recombinant allergens which can be used in the diagnostics and therapy of allergies. (Scheiner and Kraft, 1995, Allergy 50, 384-391). The preparation of recombinant Bet v 1 allergens (rBet v 1) and their purification for pharmaceutical purposes has been described, for example, by Hoffmann-Sommergruber et al. (Protein Exp. Purif. 9 (1), 1997: 33-39).
In addition, specific genetic modification of recombinant allergens is possible, enabling a reduced allergenic potential to be achieved (Schramm et al. 1999, J. Immunol. 162 (4); 2406-2414; Valenta et al., 1999, Biol. Chem. 380: 815-24; Singh et al., 1999, Int. Arch. Allergy Immunol. 119: 75-85). Allergen variants of this type are promising future candidates for specific immunotherapy of type 1 allergy.
However, a potential disadvantage in recombinant allergen variants is that the modification of the primary structure causes loss or a reduction in the reactivity of the T-cell epitopes which are necessary for therapeutic success. This possibility can only be excluded if the primary structure corresponding to the natural allergen serves as the basis for the preparation of the recombinant protein.
In the case of major birch pollen allergen Bet v 1, the preparation was carried out in two halves (Vrtala, S., et al., 1997, J. Clin. Invest. 99: 1673-81) or as a trimer (Vrtala, S., et al., 1999, Int. Arch. Allergy Immunol. 118:218-9) in order to optimise it for therapeutic purposes, i.e. to reduce the IgE binding capacity, by recombinant methods. The potential loss of T-cell epitopes and the insolubility of the proteins also has a disadvantageous effect in these approaches. A further disadvantage here can be seen in the complex manner of preparation of these rBet v 1 variants.
A suitable starting point for the preparation of a recombinant major allergen rBet v 1 which can be utilised for therapeutic purposes would accordingly be a molecule which corresponds to the wild type in the primary structure and is unrestricted in its T-cell stimulation, but has reduced IgE activity, i.e. is hypoallergenic.
This object has been achieved in accordance with the present invention by the performance of a series of biochemical purification steps known per se using soluble recombinant major allergen rBet v 1 as starting material, Surprisingly, a reduced IgE activity and at the same time maintained T-cell stimulation has been observed in the proteins purified in this way. Accordingly, the process according to the invention provides improved therapeutic efficacy at the same time as a significant reduction in or absence of side effects.
The form of the preparation process of the recombinant allergens is of particular importance here inasmuch as the proteins are converted in the course of this process into a conformation which has no or greatly reduced affinity to IgE with constant T-cell stimulation.